This invention relates to nucleic acids and encoded polypeptides of a lens epithelial cell derived growth factor (LEDGF), and diagnostics and therapeutics related to medical conditions associated with such genes and polypeptides, including cataracts.
The term cataract is used to define the opacification of the normally clear and transparent crystalline lens. Several types of cataract etiology have been described. For example, congenital cataracts occur as a complication of intrauterine rubella, herpes simplex, herpes zoster, syphilis and cytomegalic inclusion disease, the majority being idiopathic or inherited. Acquired cataracts result from trauma, radiation, drugs, metabolic disorders, ocular inflammatory disorders, or aging (senile or age-related cataracts). It has been estimated that nearly one billion elderly people throughout the world have age-related cataracts [ARCs] (Thylefors et al., Available Data on Blindness, Geneva, Switzerland; WHO Program for the Prevention of Blindness, 1994). The underlying mechanisms responsible for ARCs, however, are not well understood.
Although the anterior lens is covered with a single layer of cuboidal lens epithelial cells (LECs), most of the remaining lens is composed of anucleated fiber cells. With age, even though the lens enlarges, the epithelial cell density and cytological activity decline (Guggenmoos-Holzmann et al., Invest. Ophthalmol. Vis. Sci. 30:330-332, 1989, Konofsky et al., Ophthalmology 94:875-880, 1987, and Straatsma et al., Am. J. Ophthalmol. 112:283-296 1991); this decrease is most pronounced in lenses with cortical and mixed cataracts. Also in these cataracts, there is greater LEC metaplasia than in the normal lens (Saitoh et al., Nippon Ganka Gakkai Zasshi 94:176-180, 1990, Streeten and Eshaghian, Arch. Ophthalmol. 96:1653-1658, 1978, and von Sallmann, Am. J. Ophthalmol. 44:159-170, 1957). Since the LECs maintain lens homeostasis, a decrease in epithelial cell number or metabolic activity is expected to disturb normal lens physiology.
For survival, the cells require growth or proliferation factors, and several such factors have been reported: neurotrophic factor for neurons (Cowan et al., Science 225:1258-1264, 1984, Purves, Body and brain. A trophic theory of natural connections. Harvard University Press. Cambridge, Mass. pp. 231, 1988, Barde, Neuron 2:1525-1534, 1989, and Oppenheim, Science 240:919-922, 1991), colony stimulation factor for myeloid cells (Metcalf, Nature 339:27-30, 1989, Williams et al., Nature 343:76-78, 1990, and Koury and Bondurant, Science 248:378-381, 1990), specific hormones for endocrine-dependent cells (Kerr and Searle, Virchows. Arch. B. Cell Pathol. 13:87-92, 1973, Krypaniou and Issacs, Endocrinology 122:552-562, 1988, and Wyllie et al., J. Pathol. 111:85-94, 1973), and specific growth factors for oligodendrocytes (Barres et al., Cell 70:31-46, 1992). Ishizaki et al. predicted that LECs must have such growth factors (J. Cell Biol. 121:899-908, 1993].
It was demonstrated recently (Ibaraki et al., Exp. Eye Res. 64:229-238, 1997 and Singh et al., J. Immunol. 155:993-999, 1995), that auto-antibodies against lens P-crystallins induced LEC damage and cataract formation in mice. Both serum and monoclonal antibody (Ab) transfer studies established that humoral rather than cellular immunity was responsible for the death of LECs. It was found that anti-P crystallin antibodies (Abs) killed LECs and that damage was age-dependent (Singh et al., J. Immunol. 155:993-999, 1995). In human serum, anti-lens Abs were more prevalent in patients with age-related cataracts (98%) than in subjects with clear lenses (40%) (Singh et al., Exp. Eye Res. Submitted, 1997). It was also shown that more than 96% of the sera from patients with ARC, but fewer than 30% with clear lenses, were cytotoxic. Mixing cytotoxic anti-lens Abs with whole lens antigens (Ags) decreased or eliminated the cytotoxic effects. These findings raise the possibility that human ARCs are caused by an autoimmune insult to the LECs.
There exists a need to influence favorably the physical properties of the crystalline lens.
There also exists a need to identify the gene(s) responsible for cataract and age-related cataract in particular, and to provide therapy for preventing and treating cataracts.
An object of the invention is to provide compounds that desirably influence the physical properties of the crystalline lens.
Another object of the invention is to provide therapeutics for treating diseases or conditions involving LEDGF expression.
Still another object of the invention is to provide diagnostics and research tools relating to LEDGF. These and other objects will be described in greater detail below.
We describe herein the molecular cloning and characterization of LEDGF, a novel polypeptide that stimulates protein synthesis. This increase in protein synthesis leads to the enhanced growth of a variety of cell types such as those of epithelial, epidermal or kidney origin, including lens epithelial cells. Anti-LEDGF Abs blocked the stimulatory effects of this protein, resulting in the death of LECs and other types of cells, including neuronal, fibroblasts, etc. Increased levels of anti-LEDGF Abs were found in patients with ARCs, suggesting that neutralization of LEDGF by auto-Abs is responsible for the development of cataracts and ARCs in particular.
The invention provides isolated nucleic acid molecules, unique fragments of those molecules, expression vectors containing the foregoing, and host cells transfected with those molecules. The invention also provides isolated polypeptides and agents which bind such polypeptides, including antibodies. The foregoing can be used, inter alia, in the diagnosis or treatment of conditions characterized by the aberrant expression of a LEDGF nucleic acid or polypeptide. The invention also provides methods for identifying pharmacological agents useful in the diagnosis or treatment of such conditions. Here, we present the cDNA cloning of a 61 kDa protein, LEDGF, which stimulates proliferation of a number of different cell types, particularly those of epithelial character, and include lens epithelial cells.
According to one aspect of the invention, isolated nucleic acid molecules that code for a lens epithelial cell derived growth factor polypeptide are provided and include: (a) nucleic acid molecules which hybridize under stringent conditions to a molecule consisting of a nucleic acid of SEQ ID NO:1 and which code for a lens epithelial cell derived growth factor polypeptide, (b) deletions, additions and substitutions of (a) which code for a respective lens epithelial cell derived growth factor polypeptide, (c) nucleic acid molecules that differ from the nucleic acid molecules of (a) or (b) in codon sequence due to the degeneracy of the genetic code, and (d) complements of (a), (b) or (c). In certain embodiments, the isolated nucleic acid molecule comprises nucleotides 1-3360 of SEQ ID NO:1. In some embodiments the isolated nucleic acid molecules are those comprising the human cDNA or gene corresponding to SEQ ID NO:13. The isolated nucleic acid molecule also can comprise a molecule which encodes the polypeptide of SEQ ID NO:2.
The invention in another aspect is an isolated nucleic acid molecule selected from the group consisting of (a) a fragment of a nucleic acid molecule of SEQ ID NO:1, of sufficient length to represent a sequence unique within the human genome, and identifying a nucleic acid encoding a Lens Epithelial Cell Derived Growth Factor polypeptide, (b) complements of (a), provided that the fragment includes a sequence of contiguous nucleotides which is not identical to any sequence selected from the sequence group consisting of (1) sequences having the GenBank accession numbers of Table III, (2) complements of (1), and (3) fragments of (1) and (2).
In one embodiment the sequence of contiguous nucleotides is selected from the group consisting of (1) at least two contiguous nucleotides nonidentical to the sequence group, (2) at least three contiguous nucleotides nonidentical to the sequence group, (3) at least four contiguous nucleotides nonidentical to the sequence group, (4) at least five contiguous nucleotides nonidentical to the sequence group, (5) at least six contiguous nucleotides nonidentical to the sequence group, (6) at least seven contiguous nucleotides nonidentical to the sequence group.
In another embodiment the fragment has a size selected from the group consisting of at least: 8 nucleotides, 10 nucleotides, 12 nucleotides, 14 nucleotides, 16 nucleotides, 18 nucleotides, 20, nucleotides, 22 nucleotides, 24 nucleotides, 26 nucleotides, 28 nucleotides, 30 nucleotides, 40 nucleotides, 50 nucleotides, 75 nucleotides, 100 nucleotides, 200 nucleotides, 1000 nucleotides and every integer length therebetween.
In yet another embodiment the molecule encodes a polypeptide which, or a fragment of which, binds a human antibody.
According to another aspect, the invention involves expression vectors, and host cells transformed or transfected with such expression vectors, comprising the nucleic acid molecules described above.
According to another aspect of the invention, an isolated polypeptide is provided. The isolated polypeptide is encoded by the isolated nucleic acid molecule of claim 1, 2, or 3. In some embodiments, the isolated polypeptide is a secreted protein encoded by the nucleic acid of claim 2, comprising a polypeptide having the sequence of SEQ ID NO:2. In other embodiments, the isolated polypeptide comprises a fragment or variant of the foregoing of sufficient length to represent a sequence unique within the human genome and identifying a polypeptide that has protein synthesis induction activity, provided that the fragment includes a sequence of contiguous amino acids which is not identical to any sequence selected from the sequence group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, and SEQ ID NO:22. In another embodiment, immunogenic fragments of the polypeptide molecules described above are provided.
According to another aspect of the invention, isolated polypeptides are provided which selectively bind a polypeptide encoded by the nucleic acid of claim 1, 2, or 3. Preferably the isolated polypeptides selectively bind a polypeptide which comprises the sequence of SEQ ID NO:2, or fragments thereof (e.g. SEQ ID NO:4, SEQ ID NO:6 and SEQ ID NO:8). In preferred embodiments, the isolated binding polypeptides include antibodies and fragments of antibodies (e.g., Fab, F(ab)2, Fd and antibody fragments which include a CDR3 region which binds selectively to the LEDGF polypeptide). In certain embodiments the antibodies are human.
According to another aspect of the invention, a method is provided for determining an individual""s susceptibility to developing cataracts. A test sample containing antibodies of the individual is obtained. Anti-LEDGF antibody levels are measured in the test sample and compared to a control. The individual""s risk profile of developing a cataract is characterized based upon the level of the anti-LEDGF antibody in comparison to the control. The control may be a predetermined value such as serum titer of anti-LEDGF antibodies of OD492xe2x89xa7200. In a preferred embodiment the test sample is blood, although the test sample can also be tissue such as epidermis and buccal scrapings.
The invention also contemplates kits comprising a package including assays for anti-LEDGF antibodies, LEDGF epitopes, LEDGF nucleic acids, and instructions, and optionally related materials such as controls, for example, a number, color chart, or an epitope of the expression product of the nucleic acid of claim 1 for comparing the level of anti-LEDGF antibodies in the test sample as determined by the assay with a control value or control assay results to determine a risk of developing a cataract. The kits may also include an assay for anti-xcex2-crystallin antibodies.
According to another aspect of the invention, a method is provided for treating subjects with an abnormally elevated level of anti-LEDGF antibodies to inhibit the development of cataracts. The method involves administering to an individual in need of such treatment a polypeptide encoded by the isolated nucleic acid molecule of claim 1, 2 or 3 having protein synthesis induction activity in an amount effective to inhibit the formation of opacities in the crystalline lens. In preferred embodiments of the invention, LEDGF polypeptides are administered orally or conjugated to antibodies and administered intra venously in an amount effective to tolerize the subject to LEDGF and to lower the risk of the subject developing a cataract. Nasal administration is also particularly contemplated.
Another aspect of the invention is a method for determining the level of LEDGF expression in a subject. Expression is defined either as LEDGF mRNA expression or LEDGF polypeptide expression. Preferred embodiments of the invention include PCR and Northern blotting for measuring mRNA expression, and monoclonal or polyclonal LEDGF antisera as part of the reagents to measure LEDGF polypeptide expression. Also in certain embodiments, tissue samples such as biopsy samples, and biological fluids such as blood, are used as the source of test samples. LEDGF expression is compared to a measured control.
The invention in a further aspect involves a method for decreasing LEDGF mediated activity in a subject. An agent that selectively binds to an isolated nucleic acid molecule of the invention or an expression product thereof is administered to a subject in need of such treatment, in an amount effective to decrease LEDGF mediated activity in the subject. Preferred agents are antisense nucleic acids, including modified nucleic acids, and polypeptides such as antibodies against LEDGF.
The invention in another aspect involves a method for decreasing LEDGF mediated activity in a subject with a cancer that expresses LEDGF. An agent that selectively binds to an isolated nucleic acid molecule of the invention or an expression product thereof is administered to a subject in need of such treatment, in an amount effective to decrease LEDGF mediated activity in the subject in order to inhibit or slow down the proliferation of cancer. Preferred agents are antisense nucleic acids, including modified nucleic acids, and polypeptides such as antibodies against LEDGF.
According to another aspect of the invention, methods for inducing cell-death are also provided. The methods involve contacting a cell with an amount of an inhibitor of LEDGF effective to inhibit LEDGF mediated activity in the cell, and induce cell-death. In certain embodiments, the inhibitor is an antisense nucleic acid which inhibits the expression of LEDGF, or a polypeptide that binds to LEDGF and blocks its activity.
According to still another aspect of the invention, methods for inhibiting a cell""s differentiation both in vivo and in vitro are also provided. The methods involve contacting a mammalian cell with an amount of a LEDGF inhibitor effective to decrease LEDGF mediated activity in the mammalian cell and prevent it from differentiating. In certain embodiments, the inhibitor is an antisense nucleic acid which inhibits the expression of LEDGF, or a polypeptide that binds to LEDGF and blocks its activity.
Another aspect of the invention is a method for increasing cell proliferation. A cell expressing a LEDGF receptor is contacted with an agent that increases LEDGF stimulated cell proliferation in an amount effective to stimulate such proliferation. The agent can be a polypeptide encoded by the nucleic acid of SEQ ID NO:1. The agent can also be a polypeptide having the amino acid sequence of SEQ ID NO:2, or an LEDGF receptor stimulatory fragment thereof.
Another aspect of the invention involves a method for inducing wound healing in a subject. An agent as described in the immediately preceding paragraph is administered to a subject in need of such treatment, in an amount effective to increase LEDGF mediated activity in the subject in order to promote the proliferation of cells such as epidermal cells and accelerate wound healing.
According to still another aspect of the invention, methods for inhibiting w environmental stress-induced cell-death both in vivo and in vitro are also provided. The methods involve contacting a mammalian cell expressing a LEDGF receptor, and under environmental stress otherwise sufficient to induce cell-death, with an agent that increases LEDGF mediated activity in an amount effective to inhibit death of the mammalian cell which otherwise would result from such environmental stress. The agent can be a polypeptide encoded by the nucleic acid of SEQ ID NO:1. The agent can also be a polypeptide having the amino acid sequence of SEQ ID NO:2, or an LEDGF receptor stimulatory fragment thereof.
The methods of the invention are preferably used when increased temperatures, physical trauma, oxidative, osmotic or chemical stress, and UV irradiation form part of the environmental stress.
According to still another aspect of the invention, methods for increasing heat-shock protein activity in a cell, both in vivo and in vitro are provided. The methods involve contacting the cell with an isolated nucleic acid molecule of claim 1 or an expression product thereof, in an amount effective to increase LEDGF mediated activity in the cell and to increase heat-shock protein activity in the cell. The agent can be a polypeptide encoded by the nucleic acid of SEQ ID NO:1. The agent can also be a polypeptide having the amino acid sequence of SEQ ID NO:2, or an LEDGF receptor stimulatory fragment thereof.
The invention in another aspect provides compositions comprising an antisense nucleic acid which selectively binds to the nucleic acid of claim 1 and which reduces the expression of LEDGF, and a pharmaceutically acceptable carrier.
The present invention thus involves, in several aspects, LEDGF polypeptides, genes encoding those polypeptides, functional modifications and variants of the foregoing, useful fragments of the foregoing, as well as therapeutics and diagnostics relating thereto.
These and other objects of the invention will be described in further detail in connection with the detailed description of the invention.